In total, 40 healthy 5-week-old male C57BL/6J mice (weight, 20±2 g; SPF Biotechnology Co., Ltd.), were fed in cages and kept at 45% humidity and 20˚C under a 12-h light/dark cycle. All mice had free access to water and food. The mice were randomly assigned to the following groups: i) Sham operation group (Sham); ii) IR group + vehicle (IR + vehicle); iii) IR + SEV low-dose preconditioning (SEV-L; inhalation of 2.2% SEV); and iv) IR + SEV high-dose preconditioning (SEV-H; inhalation of 3.3% SEV). Each group contained 10 mice.

The second animal experiment aimed to further investigate the renal protective effect of SEV via activation of Nrf2. Another 40 healthy 5-week-old male C57BL/6J mice (weight, 20±2 g; SPF Biotechnology Co., Ltd.), were used (n=8 mice/group). All mice were fed in cages and kept at 45% humidity and 20˚C under a 12-h light/dark cycle, with free access to water and food. A further five groups of mice were involved: i) Sham group; ii) IR group; iii) IR + brusatol (Selleck Chemicals) group; iv) IR + SEV (high dose); and v) IR + SEV (high dose) + brusatol group. Brusatol (30 mg/kg), an Nrf2 inhibitor, was administered orally 2 h before preconditioning with SEV. Following IR, the levels of blood urea nitrogen (BUN) and serum creatinine (Scr) were detected.

The mice were anesthetized with an intraperitoneal injection of 1% sodium pentobarbital (50 mg/kg) and were fixed in the lateral position on the operating table. Longitudinal abdominal incisions from the left and right sides were cut until the kidneys were exposed. Both sides of the incision were covered with normal saline gauze. The renal pedicle capsule was peeled off and the separated renal artery on both sides was clamped with a non-invasive artery clamp. The kidney was returned in the abdominal cavity following successful clamping. The bilateral artery clamp was removed 45 min later. The kidneys were returned into their original place after confirming the recovery of blood perfusion. Subsequently, the muscle layer was sutured followed by the skin layer. All experimental animals maintained spontaneous breathing during the operation. The renal artery was not clamped in the Sham group, but all other experimental steps were the same as in the other groups. All animal experiments were conducted in strict accordance with the guidance provided on the treatment of experimental animals issued by the Ministry of Science and Technology of China (21). At the end of the experiments, all animals were euthanized by cervical dislocation.

Mice in the SEV group inhaled the mixture of low-dose or high-dose sevoflurane (Maruishi Pharmaceutical Co., Ltd.) and O₂ (80%) for 60 min, 30 min prior to IR. The renal IR model was established following anesthesia. The mice were placed in a clean environment with a constant temperature of 22˚C following surgery with free access to food and water. Urine was collected before mice were sacrificed by cervical dislocation.

The mice were sacrificed 24 h following surgery. Blood samples were extracted from the heart, placed in a 1.5 ml tube and centrifuged at 1,000 x g at 4˚C for 15 min after standing at room temperature. The supernatant was taken for further analysis. BUN and Scr were quantified using an automatic biochemical instrument (TBA-120FR; Toshiba Corporation). NGAL levels were assessed using a commercially available ELISA kit (cat. no. ab119601; Abcam). Serum inflammatory cytokine levels, including TNF-α (cat. no. ab46105), IL-6 (cat. no. ab222503), and IL-1β (cat. no. ab197742) were also measured using ELISA kits (all purchased from Abcam). Intercellular adhesion molecule-1 (ICAM-1; cat. no. ab100688), monocyte chemoattractant protein-1 (MCP-1; cat. no. ab100721) and vascular cell adhesion protein-1 (VCAM-1; cat. no. ab201278) were measured using ELISA kits (all purchased from Abcam).

Renal tissues were fixed with 4% paraformaldehyde (PFA) at room temperature for 24 h, embedded in paraffin, cut into 8-µm thick sections, and stained with H&E at 35˚C for 3 min. The pathological changes in renal tissue were observed using a light microscope. A total of two different sections were taken from each sample to observe the histopathological changes of the kidney. According to the method introduced by Zhang et al (22,23), for each animal, at least 10 high-power (magnification, x400) fields were examined. The percentage of tubules that displayed cellular necrosis, loss of brush border, cast formation, vacuolization and tubule dilation was scored as follows: 0 (none), 1 (<10%), 2 (11-25%), 3 (26-45%), 4 (46-75%) and 5 (>76%). Masson staining was performed at room temperature using a commercial staining kit (cat. no. G1340; Beijing Solarbio Science & Technology Co., Ltd.) according to the manufacturer's protocol, to evaluate collagen fibrils in renal tissues. The staining was performed according to manufacturer's instruction. A total of 10 stained fields were randomly selected from each section and observed using a CX23 RFS2 LED light microscope (magnification, x400, Olympus Corporation). The images were analyzed by Image-Pro Plus 6.0 (Media Cybernetics, Inc.). For fibrotic area semi-quantification, a ratio of blue stained area to the area of the entire field (including glomeruli, tubule lumina and blood vessels) was assessed and expressed as a percentage of Masson staining.

A total of 0.2 g kidney tissue was ground, centrifuged at 1,000 x g for 10 min at 4˚C. The supernatant was obtained to detect the concentrations of different proteins. The enzyme activity of SOD (cat. no. A001-3), myeloperoxidase (MPO; cat. no. A044) and CAT (cat. no. A007), as well as the concentration of malondialdehyde (MDA; cat. no. A003-1) were determined using commercial kits (all purchased from Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's protocol.

Immunohistochemistry was performed using a two-step method. Briefly, 5-µm thick sections of mouse kidney were dewaxed and rehydrated using xylene and gradient alcohol, treated with 1% hydrogen peroxide solution at 37˚C for 10 min, heated and repaired using an antigen repair solution (1:10; cat. no. P0088; Beyotime Institute of Biotechnology), cooled to room temperature and incubated with primary and secondary antibodies. The slide was incubated with DAB solution (cat. no. P0203; Beyotime Institute of Biotechnology) for 8 min at room temperature and then sealed for observation. Positive staining was indicated by yellowish-brown tissues. The following primary antibodies were used: Caspase-3 (1:100; cat. no. 66470-2-Ig), Nrf2 (1:100; cat. no. 66504-1-Ig), HO-1 (1: 100; cat. no. 27282-1-AP) and NQO1 (1:100; cat. no. 67240-1-Ig) (all from ProteinTech Group, Inc.). The sections were examined using a light microscope (DMLS-1000; Leica Microsystems GmbH) with a high-power field of view (magnification, x400). A total of five visual fields were randomly selected and the images were analyzed using Biosens Digital Imaging System Software version 16 (Shanghai Bio-Tech Co., Ltd.). The color of the images was transformed to separate the positive areas from the background for automatic measurement. The following formula was used: Positive target expression index (%)=average optical density of positive target x positive area/(positive area + negative area).

The kidney sections from each group were dewaxed with xylene and washed with 100% ethanol, rehydrated and washed in decreasing concentrations of ethanol and were immersed in 0.85% NaCl, then washed twice with PBS. Apoptosis was detected in tissue sections using the DeadEnd^™ Colorimetric Apoptosis Detection System (Promega Corporation). Samples were fixed with 4% PFA for 15 min at 20-25˚C and immersed in PBS twice for 10 min. Subsequently, samples were permeabilized with 20 µg/ml proteinase K solution and incubated for 20 min at room temperature. Slides were washed in PBS and fixed with 4% PFA at room temperature for 5 min, washed, and equilibrated with equilibration buffer for 10 min. Then, 100 µl terminal deoxynucleotidyl transferase mix was added to the tissue sections, which were incubated for 60 min at 37˚C and then washed in PBS. The slides were immersed in 0.3% hydrogen peroxide for 3 min and washed in PBS. Subsequently, 100 µl streptavidin HRP (cat. no. A0303; 1:500; in PBS; Beyotime Institute of Biotechnology), was added and the samples were incubated for 30 min at room temperature. After the slides were washed in PBS, 100 µl DAB solution (Beyotime Institute of Biotechnology) was added to stain the slides for 10 min at 15-25˚C. The slides were washed in deionized water and were mounted using mounting medium. TUNEL-positive apoptotic cells were visualized using a light microscope (magnification, x200).

After homogenizing the preserved kidney tissues, total protein was extracted using a protein extraction kit (Beijing Solarbio Science & Technology Co., Ltd.) and the protein concentration was quantified using the BCA method. Total protein, using 30 µg from each sample, was separated using 10% SDS-PAGE and transferred onto a PVDF membrane. The membranes were blocked with 5% skimmed milk for 1.5 h at 20-25˚C, incubated overnight at 4˚C with primary antibodies and were subsequently incubated with a suitable secondary antibody at room temperature for 1 h. An Enhanced Chemiluminescence Kit (Amersham; Cytiva) was used to detect protein bands. β-actin served as a loading control. Bands were semi-quantified via scanning densitometry using the Odyssey^® CLx Imaging System and Image Studio Version 3.1 software provided by the manufacturer (LI-COR Biosciences). The primary antibodies included caspase-3 (cat. no. 66470-2-Ig; 1:1,000), Nrf2 (cat. no. 66504-1-Ig; 1: 1,000), HO-1 (cat. no. 27282-1-AP; 1:1,000) and NQO1 (cat. no. 67240-1-Ig; 1:1,000) (all from ProteinTech Group, Inc.). HRP goat anti-mouse IgG (1:5,000; cat. no. sc-2005) or HRP goat anti-rabbit IgG (1:4,000; cat. no. sc-2054) (both from Santa Cruz Biotechnology, Inc.) was used.

The human renal proximal tubular HK-2 cell line was purchased from American Type Culture Collection. Cells were plated in 100-mm culture dishes and cultured in DMEM supplemented with 10% FBS (both from Thermo Fisher Scientific, Inc.), 100 U/ml penicillin, 2 mM glutamine, 100 µg/ml streptomycin and 1 mM HEPES buffer. Cells were cultured at 37˚C in humidified air containing 5% CO₂. The medium was replaced every other day. For the H/R group, cells were exposed to hypoxic conditions (5% CO₂, 1% O₂ and 94% N₂) for 24 h followed by 12 h of reoxygenation (5% CO₂, 21% O₂ and 74% N₂). To determine the effect of SEV, cells were pretreated with either vehicle buffer (0.5% DMSO solution) or SEV (1 and 2 concentration) for 12 h prior to H/R treatment. A Nrf2 inhibitor, brusatol (10 nM) was also added to evaluate whether SEV, serving its protective role, was dependent on the Nrf2 signaling pathway. The in vitro study contained the following six groups; i) Control group without H/R; ii) H/R group treated with vehicle; iii) H/R group treated with 1% SEV; iv) H/R group treated with 2% SEV; v) H/R group treated with brusatol; and vi) H/R group treated with brusatol and 2% SEV. An in-line anesthetic vaporizer (Drägerwerk AG & Co., KGaA) fed by a supply of a gas containing 21% O₂ and 5% CO₂, balanced with N₂, was used to deliver SEV at a rate of 2 l/min for at least 5 min until the desired SEV concentration (1-2%) was achieved. Concentrations of SEV and O₂ were monitored every h using an anesthetic analyzer (Drägerwerk AG & Co. KGaA). Cells in the vehicle control conditions were placed in an identical gas chamber under 21% O₂ and 5% CO₂, balanced with N₂. Once sealed, the chambers were placed in a 37˚C incubator.

HK-2 cells were treated with Cell Counting Kit-8 reagent (10 µl/well; Applygen Technologies, Inc.) for 2 h. The optical density was recorded at 450 nm using a microplate absorbance reader (Tecan Group, Ltd.).

The data were analyzed using GraphPad Prism 8.0 software (GraphPad Software, Inc.). Data are presented as the mean ± SD. Statistical comparisons among the groups were analyzed using one-way ANOVA followed by Bonferroni's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Compared with that in sham group, the concentration of BUN, Scr, and serum NGAL in the IR group were significantly increased (all P<0.01) (Fig. 1A). Preconditioning with SEV decreased the BUN, Scr and serum NGAL concentrations in a dose-dependent manner (Fig. 1A). The H&E staining (Fig. 1B) also demonstrated that preconditioning with SEV attenuated acute renal tubular injury induced by I/R in a dose-dependent manner, consistent with the biochemical analysis.

It was determined via TUNEL staining that preconditioning with SEV resulted in a significantly reduced quantity of apoptotic tubular cells in the renal interstitium compared with that in the vehicle control group (P<0.05; Fig. 2A). Actively cleaved caspase-3 protein was mainly expressed in the cytoplasm and nucleus of impaired renal tubular epithelial cells. The results demonstrated that the protein expression level of cleaved caspase-3 in the sham group was low, whereas IR-induced AKI increased the caspase-3 expression levels in the cytoplasm and nucleus of renal tubular epithelial cells. SEV preconditioning significantly decreased the caspase-3 protein expression levels in a dose-dependent manner, especially in the SEV-H group (Fig. 2B). Western blotting (Fig. 2C) also revealed that SEV preconditioning reduced cleaved caspase-3 protein expression levels compared with that in the IR vehicle group (P<0.01).

The results demonstrated that IR led to the increased levels of cytokines and chemokines, including TNF-α, IL-6, IL-1β, ICAM-1, MCP-1 and VCAM-1 compared with in the sham group (Fig. 3). SEV preconditioning significantly reduced cytokine and chemokine levels in the peripheral blood compared with in the IR + vehicle group, especially in the SEV-H group. Similarly, SEV preconditioning significantly decreased concentrations of MDA and MPO, whereas the activity of SOD and CAT was significantly increased as shown in Fig. 4.

Due to the antioxidant properties of SEV preconditioning as demonstrated in the aforementioned results, it was hypothesized that SEV preconditioning might activate the endogenous antioxidant defense system and that the Nrf2 signaling pathway was a possible target. By performing immunohistochemistry it was determined that the Nrf2 protein expression levels in kidney tissue in the preconditioning groups were notably increased compared with that in the IR group. Moreover, its downstream proteins HO-1 and NQO-1 were also notably upregulated by SEV preconditioning (Fig. 5A). Western blotting demonstrated a similar trend to the immunohistochemistry results (Fig. 5B), which further confirmed that SEV preconditioning may activate the Nrf2 signaling pathway in the kidneys from the mouse model of IR.

In clinical practice, IR may cause renal interstitial fibrosis in the later stages and consequently, may affect the prognosis of AKI. A similar situation was discovered in animal models (24). In the present study, IR resulted in significant fibrosis of the renal interstitium in mice two weeks after IR, which was assessed using the ratio of Masson staining in high resolution fields (Fig. 6). As well as fibrosis, widening of the renal interstitium and atrophy of renal tubules were also evident in the microscopic sections. The degree of fibrosis was significantly reduced following SEV preconditioning, as well as amelioration of widening of the renal interstitium and atrophy of renal tubules.

To further confirm the renal protective effect of SEV via the activation of Nrf2, another five groups of animal experiments were conducted. Brusatol (30 mg/kg), an Nrf2 inhibitor, was orally administered 2 h prior to SEV preconditioning to the allocated groups. Following IR, the BUN and Scr levels were detected. The results demonstrated that brusatol administration slightly aggravated renal injury in IR mice (Fig. 7A). Moreover, the renal protective effect of SEV was almost abolished by pretreatment with brusatol. The Nrf2 protein expression levels in kidney tissues were significantly reduced by Brusatol treatment as shown in Fig. 7B. Similar results were obtained using the in vitro HK-2 H/R cell model. The results demonstrated that H/R treatment markedly reduced HK-2 cell viability. SEV pretreatment increased cell viability in a dose-dependent manner, however, its protective effect was significantly reduced when the cells were also treated with brusatol (Fig. 7C). Western blot also confirmed that Nrf2 protein expression level in HK-2 cells was markedly reduced by brusatol treatment (Fig. 7D). These results indicated that SEV may exert renal protection, which is dependent on the activation of Nrf2.